インドネシアの魚類とアコヤガイ養殖場における病魚と健康魚からのウイルス検出

誌名誌名愛媛大学農学部紀要 = Memoirs of the College of Agriculture, EhimeUniversity

ISSNISSN 04246829

著者著者

北村, 真一金平, 圭介武岡, 英隆鈴木, 聡

巻/号巻/号 52巻

掲載ページ掲載ページ p. 9-11

発行年月発行年月 2007年9月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

愛媛大学農学部紀要 (Mem.Fac. Agr.， Ehime Univ.l

52: 9 -11 (2007)

9

Virus Detection in Diseased and Healthy Fish and Oysters

from an Indonesian Aquaculture Site: Double Infection of Viral Nervous

Necrosis Virus (VNNV) and Marine Birnavirus (MABV)

Shin-Ichi KITAMURAぺKeisukeKANEHIRAぺHidetaJくaTAKEOKAキ andSatoru SUZUKIホホ

Abstract

In the south Sumatra region of Indonesia， tiger group巴r，Epinephelus juscoguflatus， and humpbuck grouper，

Cromileplesαltivelis， ar巴 commerciallyimportant fish sp巴cies.Recently， however， mass mortalities of the two species

have occurred in the region and th巴 causeof th巴S巴 mortalitieshas not been c1arified. We surveyed the distribution of

fish virus巴sincluding viral nervous necrosis virus (YNNY)， marine birnavirus (MABY) and red seabream iridovirus

(RSIY) in the two grouper species and MABV in silver th巴 lipoyster， Pinctada maxima， by PCR. YNNY was detected

from both healthy and diseased group巴rswhereas MABY was only detected in diseased groupers， sugg巴stingthat the

mass mortalities of the two grouper species wer巴causedby co-infection of YNNY and MABY. PCR product of RSTY

was not observed in any fish salllples. In addition， MABV was not detected in silver lip oysters frolll both healthy and

those exhibiting poor growth

Key words: Tiger grouper， humpbuck grouper， lip oyster， YNNY， MABY， lndonesia

Introduction

Marine aquaculture industries including fish and

shel¥fish sp巴cieshave been developed in lndonesia. In th巴

south Sumatra region of lndonesia， tiger grouper，

Epinephelus fuscoguffatus， and humpbuck grouper，

Cromileptes alfivelis， ar巴 commercial¥y important fish

species， however disease outbreaks due to unknown

causes have been 巴ncount巴red in these fish species，

resulting in a serious economic loss to th巴 aquaculture

industry. ln shel¥fish sp巴ci巴s， the silver Iip oyster，

Pinctada maxuna， is one of the Illost important cultured

sh巴I¥fish species to produce pearls in the region， but

ventures in shel¥fish aquaculture have also faced problems

such as poor growth of the oyster due to unknown

reasons

From the viewpoint of infectious disease， viral diseases

have often caused Illass mortality of cultured fish and

shel¥fish species. For example， viral nervous necrosis virus

(VNNV) belonging to genus 8etanodavirus in family

Nodaviridae (Muroga， 2001)， red seabream iridovirus

(RSIY) belonging to the genus Megalocytivirus in family

Iridoviridae (lnouye et al. 1992) and marine birnavirus

(MABV) belonging to genus Aquabirnavirus in family

8irnaviridae (Sorimachi & Hara， 1985) were d巴tected

Received 27 March 2007. Accepted 22 June 2007 ホCent巴rfor Marine Environlllental Studies，日1imeUniversity，

Bunkyo 3， Matsuyallla 790-8577， Ehillle， Japan (**Co汀巴-

sponding author)

from Illany diseased fish species in Asian countries. To

control viral diseases， it is n巴cessary to understand

distributions of the fish viruses in cultured fish and

shel¥fish species as a basic research， but it has not b巴巴n

ful¥y investigated in lndonesia so far. Therefor巴， th巴

distribution of above three viruses in the tiger group巴rand

humpback grouper， and silver Iip oyster cultured in south

SUlllatra region of lndonesia was surveyed including

exallline the cause of mortalities in two grouper fish

species in this study.

九1aterialsand民1ethods

Fish and shelIfish

Healthy and diseased tiger grouper and humpbuck

grouper were sampled from an aquaculture facility in

Hurun Cov巴 insouth Sumatra of Indonesia in July 2003

(Fig. 1， Station 5). Diseased fish show巴d sluggish

behavior n巴arthe water surface and resting on the bottom，

and exhibited a mortality rate of >30%. Healthy and poor-

growing silver lip oyst巴rswere col¥ected from an oyster

farm managed by a local privat巴 cOlllpany(Fig. l， Station

27).

Detection of fish viruses by PCR

Spleen and brain of fish were pooled and used for

extraction of nucleic acids. ln the case of shellfish，

h巴patopancreas and hemocyte of the oyster were

separately collected to detect MABV becaus巴 thevirus

could potential¥y infect both tissues in the Japanese pearl

oyster Pincfada fucata (Kitamura et al. 2000). Total

nucleic acids were extracted from the col¥ected samples

10 Shin-[chi KITAMURA， Keisuke KANEHIRA， Hidetaka TAKEOKA and Satoru SUZUKI

10S'14'E 10告。16'E 105'17・E IOS'18'E

5・3日'S

‘~ -:':lL 5'3，'S

--・"~-105.0 110.0 ll!i.O 120.0 l2!5.0

Lon&itude

S四 101'50.000

Fig.l. Map of sampling stations. .; sampling station of tiger grouper and humpbuck grouper， .; sampling station of silver lip oyster.

using the method d巴scribed by Suzuki et al. (1997).

Briefly， th巴 organwas homogenized in 4-fold volume of

TE (0.2 M Tris HCI， pH 8.0， 0.1九1EDT A)， and was

c巴ntrifuged at 3，000 x g for 5 min. Th巴 45μ1 of

supernatant was taken and 5μ1 of proteinas巴 K(lmg/ml;

TaKaRa Co.， Shiga， Japan) was added. The mixture was

incubated at 55 oC for 2 h. NucJeic acids were extracted

using phenol-chloroform. A portion of the nucJeic acids

were sl1bjected to reverse transcription (RT) for VNNV

and MABV d巴tection，describ巴d by Nishizawa et al.

(1994) and Suzuki et al. (1997)， respectively; PCR was

then performed. In order to detect RSIV， the remaining

nl1cJeic acids were directly used for PCR by the method

of Kurita et al. (1998). PCR amplification was performed

with a DNA th巴rmalcycJer (TaKaRa Co.， Shiga， Japan).

Amplified prodllcts were analyzed by agarose gel

electrophoresis， and were visllalized with ethidillm

bromide and UV light irradiation.

Results and Discussion

ln fish samples collected from the south Sumatra region

of Indonesia， VNNV was detected from both h巴althyand

diseased tiger and humpbuck grollper. The detection rate

of VNNV was 100% from diseased individllals of both

species， whereas it was <66.6% in what w巴redesignated

as healthy fish (Table 1). Since Zafran et al. (2000)

detected the same virus from dis巴asedjllv巴nilehllmpbllck

Tabl巴 1.PCR detection of thre巴 fishviruses from tiger grol1p巴r，humpbuck grouper and oyster

Species Condition Fish no. mean B.W. (g) PCR positiv巴 rate(%)

YNNY 恥1ABY RSIY

Tiger grol1per Healthy 3 41.4 33.3 O O

Tiger grouper Diseased 3 52.2 100 100 O

Hl1mpbllck grol1per H巴althy 3 28.9 66.6 O O

Humpbuck grouper Diseased 3 36.7 100 66.6 O

Silver lip (Hepatopancr巴as) Healthy 10 49.9 NT持 O NT Silver lip (Hemocyte) Healthy 10 NT 。 NT Silver lip (Hepatopancreas) Poor growing 10 26.9 NT O NT Silver lip (Hemocyte) Poor growing 10 NT O NT

ドNottested

Virus Detection in Diseased and Healthy Fish and Oysters from an Indonesian Aquaculture Site: Double Infection of Viral Nervous Necrosis Virus (VNNV) and Marine 8imavirus (MA8V) 11

grouper in the Bali r巴gion of Indonesia， the virus is

thought to be widely distributed in the country. In the

pres巴nt study， symptoms of a diseas巴d fish， sluggish

behavior and resting on the bottom， was similar to that

observed for fish affected with VNNV， suggesting that the

main cause of mortality in the two grouper species is

VNNV infection. RSIV was not detected from any of the

grouper samples. Since the virus has caused mass

mortality in many fish species， incIuding grouper， many

South巳astAsian countries requir巴sustam巴dsurveillance of

the virus (Miyata et al. 1997; Sudthongkong et al. 2002;

Gibson-Kueh et al. 2004).

Interestingly， the high detection rate (>66%) of MABV

was obs巴rvedin only diseas巴dgroupers， indicating that

co-infection by VNNV and MABV occurred in the fish.

Regarding the co-infection by bimavirus and the other

fish virus， from experiemental infection of Japanese

f10under (Paralichthys o/ivaceus). Pakingking et al.

(2003) reported that primary aquabimavirus (ABV)

infection suppressed the secondary viral hemorrhagic

septicemia virus (VHS) infection. However， our

observations suggest that MABV enhanced the

pathogenicity of VNNV because MABV was det巴ct巴d

from only diseased individuals of grouper， despite VNNV

having been detected from both healthy and diseased fish.

To cIarify the re¥ationship between mortality and co-

infl巴ctionof th巴seviruses in grouper， th巴巴xperimentalco-

infection in consideration of the contagious tum of VNNV

and MABV is necessary.

Altematively， in the single-infection of bimavirus in

grouper (Epinephelus sp.) fry， the pathogenicity of the

virus was low in巴xperimentalinfections， but it increas巴d

when fish were exposed to a stressor such as heavy metals

and changes in salinity (Chou et al. 1999). This evidence

suggests that the virus is an opportunistic pathogen in

grouper and its pathogenicity could be enhanced by

environmental pollution and physiological factors.

Recently， high population densities of cultured fish

coupled with environmental pollution have created ideal

conditions for infectious diseases， making it a serious

problem in Asian countries. Therefore， monitoring of

Cultured fish species in Indonesia for opportunistic

pathogens such as MABV is also highly warranted.

In shellfish samples of silver lipped oysters， MABV

was not detected from any individuals (i.e. poor or regular

growth)， and th巴refl

Acknowledgements

We thank staff of Agency for the Assessm巴nt and

Application Technology (BPPT) and National Seafarming

Development Center (BBL) for their he¥p in fish and

shellfish sampling. This work was partly support巴dby a

Grant-in-Aid for Scientific Research (Pr句ect No.

l4254004)， Japanese Society for the Promotion of

Sciences (JSPS)， and 21" Century COE program. Dr.

Todd Miller is appreciated for his critical reading of this

paper.

References

Chou HY， Peng TY， Chang SJ， Hsu YL， Wu JL (1999) Effect

of heavy metal stressors and salinity shock on the

susceptibility of grouper (Epinephelus sp.) to infectious

pancreatic necrosis virus. Yirus Res 63: 121-129

Gibson-Kueh S， Ngoh-Lim GH， N巴ttoP， Kurita J， Nakajima

K， Ng ML (2004) A systemic iridoviral diseas巴 inmullet，

Mugil cephalus L.， and tiger grouper， Epinephelus

fuscoguttatus Forsskal: a first r巴portand study. J Fish Dis

27: 693-699

lnouye K， Yamano K， Maeno Y， Nak勾imaK， Matsuoka M，

Wada Y， Sorimachi M (1992) lridovirus infection of

cultured red s巴abream， Pagrus major. Fish Pathol 27: 19-

27.

Kitamura Sl， Jung SJ， Suzuki S (2000) Seasonal chang巴 of

infective state of marine birnavirus in Japanese pearl oyster

Pinctada fucαta. Arch Yirol 145: 2003-2014

Kurita J， Nakajima K， Hirono 1， Aoki T (1998) Polymerase

chain reaction (PCR) amplification of DNA of r巴d sea

bream iridovirus (RSIY). Fish Pathol 33: 17 -23

Miyata M， Matusno K， Jung SJ， Danayadol Y， Miyazaki T

(1997) Genetic similarity of iridoviruses from Japan and

Thailand. J Fish Dis 20: 127 -134

Muroga K (200 1) Yiral and bacterial dis巴a児 sof marine fish

and shellfish in Japanese hatch巴ries.Aquaculture 202: 23

44

Nishizawa T， Mori K， Nakai T， Furusawa 1， Muroga K (1994)

Polymerase chain reaction (PCR) amplification of RNA of

striped jack nervous necrosis virus (SJNNY). Dis Aquat

Org 18: 103一107

Pakingking Jr R， Takano R， Nishizawa T， Mori K， lida Y，

Arimoto M， Muroga K (2003) Experimental coinfection

with aquabirnavirus and viral hemorrhageic septic巴mlavlrus

(YHSY)， Edwardsiella tarda or Streptococcus iniae in

Japan巴seFlounder Paralichthys olivaceus. Fish Pathol 38:

15-21

Sorimachi M， Hara T (1985) Characteristics and pathogenicity

of virus isolated from yellowtail fingerlings showing ascites.

Fish Pathol 19: 231 -238

Sudthongkong C， Miyata M， Miyazaki T (2002) Yiral DNA

sequences of genes encoding the ATPase and the major

capsid protein of tropical iridovirus isolates which are

pathogenic to fishes in Japan， South China Sea and

Southeast Asian countries. Arch Yirol 147: 2089 -2109

Suzuki S， Hosono N， Kusuda R (1997) D巴tectionof aquatic

birnavirus gene from marine fish using a combination of

r巴V巴rsetranscription-and n巴stedPCR. J Mar Biotechnol 5:

205-209

Zafran 1， Koesharyani FJ， Yuasa K， Harada T， Hatai K (2000)

Yiral n巴rvousnecrosis in humpback group巴rCrO/ηileptes

altivelis larvae and juveniles in lndonesia. Fish Patho